Retaining microfluidic microcavity and other microfluidic structures

ABSTRACT

A microfluidic device that comprises several microchannel structures in which there are an inlet port, an outlet port and there between a structural unit comprising a fluidic function. The structural unit can be selected amongst units enabling a) retaining of nl-aliquots comprising constituents which has been defined by mixing of aliquots within the microfluidic device (unit A), b) mixing of aliquots of liquids (unit B), c) partition of larger aliquots of liquids into smaller aliquots of liquids and distributing the latter individually and in parallel to different microchannel structure of the same microfluidic device (unit C), d) quick penetration into a microchannel structure of an aliquot of a liquid dispensed to an inlet port of a microchannel structure (unit D), and e) volume definition integrated within a microchannel structure (unit E).

This application is a continuation of U.S. application Ser. No.10/229,676, filed Aug. 28, 2002 now U.S. Pat. No. 6,919,058, whichclaims priority to U.S. Provisional Application No. 60/315,471, whichwas filed on Aug. 28, 2001; U.S. Provisional Application No. 60/322,621,which was filed on Sep. 17, 2001; U.S. Provisional Application No.60/376,776, which was filed on Apr. 30, 2002; International ApplicationPCT/SE02/00531, which was filed on Mar. 19, 2002; InternationalApplication PCT/SE02/00537, which was filed on Mar. 19, 2002; U.S.application Ser. No. 10/148,083, which is the National Stage ofInternational Application PCT/SE02/00538 filed on Mar. 19, 2002; andU.S. application Ser. No. 10/148,084, which is the National Stage ofInternational Application PCT/SE02/00539 filed on Mar. 19, 2002, and isa continuation-in-part of U.S. application Ser. No. 10/004,424 filed onDec. 6, 2001, Swedish Application Nos. 0104077-3 filed Dec. 5, 2001,0103522-9 filed on Oct. 21, 2001, and 0201310-0 filed Apr. 30, 2002which are all incorporated herein by reference.

BACKGROUND OF THE INVENTION

I. Field of Invention

The present invention concerns a microfluidic device in which there is amicrochannel structure which comprises (a) one or more inlet ports, (b)one or more outlet ports, and (c) a structural unit which comprises afluidic function and is located between one of the inlet ports and oneof the outlet ports. The structural unit (c) may include an inlet or anoutlet port.

According to the invention the structural unit is selected by certaininnovative structures permitting a) retaining nl-aliquots of liquids inwhich the constituents have been defined by mixing of aliquots withinthe microfluidic device (unit A), b) mixing of aliquots of liquids (unitB), c) partition of larger aliquots of liquids into smaller aliquots ofliquids and distributing the latter individually and in parallel todifferent microchannel structure of the same microfluidic device (unitC), d) quick penetration into a microchannel structure of an aliquot ofa liquid dispensed to an inlet port of a microchannel structures (unitD), and e) volume definition integrated within a microchannel structure(unit E). There may in addition also be other structural units and/ormicrofluidic functionalities included.

II. Related Art

Microfluidic structures have been considered promising for assays,chemical synthesis etc. which are to be performed with a high degree ofparallelity. A generally expressed desire has been to run the completesequence of steps of test protocols, including sample treatment withinmicrofluidic devices. This has lead to a desire to dense-packmicrochannel structures on planar substrates (chips) and to integratevalve functions, separation functions, means for moving liquids etc.within microfluidic devices. In the macroscopic world these kinds offunctionalities can easily be integrated into various kinds of liquidtransportation systems, but in the microscopic world it has becomeexpensive, unreliable etc. to miniaturize the macroscopic designs. Thesituation becomes still worse when moving from μl- to nl-aliquots orfrom microchannel dimensions of above 100 μm down to those less than 100μm. One of the main reasons for this is that the surface forces ofliquids are more influential on liquid behavior when going down involume from the μl-volumes to the nl volumes and smaller, for instancewhen going below 5 μl. A typically example is that wicking/imbibing willpromote quick liquid transport from a nl-vessel making it difficult toretain a specified liquid volume in such a vessel.

I. Centrifugal Force for Moving Liquids in Microfluidic Devices

The use of centrifugal force for moving liquids within microfluidicsystems has been described for instance by Abaxis Inc (WO 9533986, WO9506870, U.S. Pat. No. 5,472,603); Molecular devices (U.S. Pat. No.5,160,702); Gamera Biosciences/Tecan (WO 9721090, WO 9807019, WO9853311), WO 01877486, WO 0187487; Gyros AB/Amersham Pharmacia Biotech(WO 9955827, WO 9958245, WO 0025921, WO 0040750, WO 0056808, WO 0062042,WO 0102737, WO 0146465, WO 0147637, WO 0147638, WO 0154810, WO 0241997,WO 0241998, WO 2002074438, WO 2002075312, WO 200275775, WO 2002075776).See also presentations made by Gyros AB at various scientific meetings:(1) High-through put screening SNP scoring in microfabricated device.Nigel Tooke (September 1999); (2) Microfluidics in a rotating CD(Ekstrand et al) MicroTAS 2000, Enschede, The Netherlands, May 14-18,2000. (3) (a) SNP scoring in a disposable microfabricated CD device(Eckersten et al) and (b) SNP scoring in a disposable microfabricated CDdevice combined with solid phase Pyrosequencing™ (Tooke et al) HumanGenome Meeting, HGM 2000, Vancouver, Canada, Apr. 9-12, 2000, (4)Integrated sample preparation and MALDI MS on a microfluidic compactdisc (CD with improved sensitivity (Magnus Gustavsson et al) ASMS 2001(spring 2001).

II. Unit A: Retaining Microcavity for nl-Aliquots

The proprietor of the present invention has during the last yeardeveloped microfluidic systems comprising structural units comprisingmicrocavities intended for nl-volumes of liquids. See for instance WO9955827, WO 9958245; WO 0040750, WO 0146465, WO 0147638, WO 0241997, andWO 0241997 and scientific presentations made by Gyros AB (see above).Hydrophobic surface breaks for preventing undesired creeping of liquidaround corners or as valves have in particular been emphasized in WO9958245. See also WO 2002074438, WO 2002075312, WO 2002075775 and WO2002075776.

III. Unit B: Mixing Unit

Units for mixing aliquots within microfluidic devices have previouslybeen described. These units have been based on (a) mechanical mixers inmixing microcavities or microconduits including creation of turbulenceby fixed streric hinders (e.g., WO 9721090 and U.S. Pat. No. 4,279,862(Bretaudiere et al)); (b) creation of turbulent flow in a microcavity bytwo incoming liquid flows (e.g., WO 9853311); (c) creation of a laminarflow in the inlet end of a mixing microconduit and achieving mixing bydiffusion during the transport through the microconduit (e.g., U.S. Pat.No. 5,637,469, (Wilding & Kricka); (d) mixing by pumping layeredaliquots back and forth in a mixing microcavity or microconduit. Thiscan be accomplished by applying pulsed centrifugal force by spin pulsesthat drive the liquid in one direction and a higher spin pulse and inthe reverse direction at a lower spin pulse utilizing energy built up inthe system during a high pulse for driving the liquid in the reversedirection during a lower spin pulses. This can be accomplished byutilizing enclosed air ballast chambers and/or hydrophobic/hydrophilicas outlined in WO 0187487. The principle of back and forth transport isalso described in WO 2002074438 (unit 5) and WO 9958245.

IV. Unit C: Distribution Manifold

According to the inventors knowledge publications related to this topicare rare. U.S. Pat. No. 6,117,396 (Orchid) gives a non-centrifugalgravity based microfluidic device in which a common reagent channel isused both as an overflow channel and as a reagent fill channel. Aplurality of parallel volume metering capillaries is connected atdifferent positions to the reagent fill channel from below. Acentrifugally based distribution manifold for microfluidic systems hasbeen given in WO 9958245 and WO 0187486. This latter variant is based onan annular distribution microconduit and comprises at least onewaste/overflow microconduit per aliquot to be dispensed.

Microfluidic devices with a microchannel structures that comprises apart that bents towards a lower level (downward bent) and/or a part thatbents towards a higher level (upward bent) have been describedpreviously. Downward and upward bents have been linked to each other inshort series. Bent structures for centrifugal based system have beenused for metering liquids, process chambers etc.

Downward bents have been combined with centrifugal force and used forretaining liquid (valve function) that is to be subjected to distinctprocess steps in the bent, e.g., chemical or biochemical reactions,affinity reactions, measurement operations, volume metering etc. Byincluding an outlet microconduit with a valve function, for instance apassive valve, in the lower part of the bent, processed aliquots canbeen transported further downstream in the structure in a controlledmanner.

Further details about previously known bent structure are given in: WO9958245; WO 0147638; WO 0146465; WO 0040750; WO 2002074438, WO2002075312, WO 2002075775 and WO 2002075776; WO 0241997 and WO 0241998.Bent structures have also been indicated in scientific presentationsmade by Gyros AB and given elsewhere in this specification.

V. Unit D: Inlet Port

Imbibing has been utilized to promote liquid penetration intomicrochannel structures by including edge/corner structures associatedwith inlet ports. See U.S. Pat. No. 4,233,029 (Eastman Kodak) and U.S.Pat. No. 4,254,083 (Eastman Kodak).

VI. Unit E: Integrated Volume-Defining Unit

Integrated volume defining units in microfluidic systems are previouslyknown. U.S. Pat. No. 6,117,396 (Orchid), for instance, gives anon-centrifugal gravity based system in which a common reagent channelmay act as an overflow/filling channel along which there is spaced aplurality of volume metering capillaries for μl-volumes. Integratedunits for metering volumes in centrifugal based system by the use of anoverflow channel have been described in WO 9853311, WO 0146465 and WO0040750.

The present invention is the first to provide novel fluidicfunctionalities that are used when transporting and processingnl-volumes of liquids in microchannel systems, which are defined herein.

BRIEF SUMMARY OF THE INVENTION

The microchannel structures of the present invention are intended fortransport and processing of one or more aliquots of liquids (thus thedevice is named microfluidic). In preferred variants capillary force andcentrifugal force are used for the transport of the aliquots. Theinvention also concerns various methods in which the microfluidicdevice/microchannel structures is/are used.

In one first aspect, the invention relates to the microfluidic device asgenerally defined herein. A characteristic feature of this aspect of theinvention is that at least one of the structural units is selectedamongst the innovative units A-E described below. Units that combine thefunctionality and/or structure of two or more of the units A-E may beincluded. Other units that are known or will be known in the future mayalso be included as long as at least one of the innovative units A-E ispresent. For additional units see also PCT/SE02/00531.

In preferred variants of this aspect at least one of the aliquotsreferred to in the description of a structural unit should have asurface tension, which is ≧5 mN/m, such as ≧10 mN/m or ≧20 mN/m.

A second aspect of the invention is a method for transporting one, twoor more aliquots through a microchannel structure of the microfluidicdevice. The method comprises the steps of (i) providing the microfluidicdevice, (ii) providing said one, two or more aliquots, (iii) introducingeach of said aliquots through an inlet port of one, two or moremicrochannel structures of the device, (iv) transporting the aliquotsthrough at least one of the structural units which is present between aninlet port and an outlet port without utilizing valves and pumpscontaining movable mechanical parts, and (v) possibly collecting thealiquots in treated form in one or more of the outlet ports of themicrochannel structure. Preferable aspects include that one, two, threeor more of the aliquots that are to be introduced through an inlet portof the microchannel structures have a surface tension which is ≧5 mN/m,such as ≧10 mN/m or ≧20 mN/m.

The microfluidic device provided in step (i) is according to the firstaspect. In step (ii), at least one of the aliquots has a volume in thenano-liter range. In step (iii) two or more of the aliquots may beintroduced via the same or different inlet ports. In step (iv) thedriving force utilized for transport of the aliquots typically iscapillary force and/or inertia force without excluding other kinds offorces as discussed elsewhere in this specification.

Steps (iii) and (iv) include that the various aliquots are processedaccording to an intended protocol, i.e., the transport step (step (iv))includes that an aliquot introduced into a microchannel structure may betransported to a certain position (structural unit) and/or processed ina predetermined manner before the next aliquot is introduced. The partsequence that comprises steps (iii) and (iv) may thus be interrupted fordispensation steps, process steps etc. to take place, be divided insubsteps. For instance, The two reactants may be dispensed separately insequence to the same or to different inlet ports and then mixed in aseparate mixing unit as discussed elsewhere in this specification.Subsequent to the mixing the reaction mixture is transported to areaction microcavity and retained therein while the reaction is allowedto proceed according to the desired protocol, after which the result ofthe reaction is analyzed in the same microcavity or further downstreamor outside the microchannel structure. The analysis may involvedetermination/detection of products and/or the disappearance of one ormore of the reactants. By properly designing the system the proceedingof the reactions may be followed through the wall of the microcavity,i.e., there may also be substeps run in parallel (measuring andincubation).

In step (v) the term “treated form” contemplates that the aliquots havepassed the structure and been subjected to one or more predeterminedtreatments. The chemical composition may have changed and/or aliquotsmay have been mixed.

At least one of the aliquots is typically aqueous and/or may contain oneor more surface-active agents that increase or decrease the surfacetension of a liquid, such as water. Typical agents that reduce surfacetension are detergents that may be cationic, anionic, amphoteric ornon-ionizable. Surface-active agents include organic solvents,preferably miscible with water. Examples are methanol, ethanol,isopropanol, formamide, acetonitrile etc. Charged or chargeablepolymers, biomolecules such as proteins, certain sugars etc. may alsoact as surface-active agents.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawing, in which:

FIG. 1 illustrates the definitions of “edge” and “circumferential zone”;

FIG. 2A-FIG. 2 e illustrate innovative variants of units A and B;

FIG. 3A-FIG. 3C illustrate innovative variants of unit C;

FIG. 4A-FIG. 4B illustrate innovative variants of unit D.

FIG. 5 illustrates an innovative variant of unit E.

The structural units are viewed from above. The cross-sectional areas ofthe microconduits and microcavities are typically rectangular. Thedepths of the microchannel structures shown are typically constant andwithin the interval 100-150 μm. The widths for liquid transportmicroconduits are typically within the interval 100-300 μm and for airmicroconduits within the interval of 40-100 μm. Also compare the figuresin WO 2002074438, WO2002075312, WO 2002075775 and WO 2002075776 (all ofGyros AB), which disclose other structures of comparable dimensions.FIGS. 2 c-d specifically include certain dimensions in μm. Circlesrepresent openings to ambient atmospheres (inlet port, outlet ports,vents etc.).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel fluidic functionalities that can beused when transporting and processing nl-volumes of liquids inmicrochannel systems of the kind defined herein. A particular intentionis to create functionalities that do not require movable mechanicalparts, e.g., to accomplish valving, pumping, mixing etc., and can beintegrated into the microchannels and/or the substrates. The variousnovel functionalities are based on local surface characteristics of theinner walls of the microchannels and/or on properties of the liquids,such as surface tension and wetting ability.

A first object is to provide a structural unit with a functionality thatpermits retaining of a defined nl-aliquot for prolonged period of timein a predetermined microcavity (retaining microcavity) of a microchannelstructure of a microfluidic device. The nl-aliquots concerned areobtained by mixing nl-aliquots within the microchannel structure. Yetfurther, the term “aliquot” refers to an aliquot of a liquid if nototherwise specified. The term “prolonged” typically means that theliquid is retained in the retaining microcavity under static conditions,i.e., non-flow conditions. The period of time concerned is typically ≧15seconds, such as 30 seconds or ≧1 minute, such as ≧5 minutes or ≧10minutes, for instance ≧1 hours or ≧10 hours. The contemplated periodslast typically ≦24 hours such as ≦12 hours. This object primarily aimsat minimizing liquid losses due to wicking and/or evaporation fromretaining microcavities with volumes in the nl-range during incubationsfor performing reactions and measurements in the mixed nl-aliquot, forstorage purposes etc. Acceptable losses are typically ≦20%, such as ≦10%or ≦5%.

A second object is to provide a structural unit with a functionalitythat permits simple, quick, safe, reproducible and reliable mixing oftwo aliquots that are miscible with each other within a microchannelstructure of a microfluidic device.

A third object is to provide a structural unit with a functionality thatpermits simplified and reliable distribution in parallel to separatesubstructures of a plurality of microchannel structures in amicrofluidic device.

A fourth object is to provide a structural unit with a functionalitythat facilitates rapid introduction of an aliquot into a microchannelstructure of a microfluidic device.

A fifth object is to provide a structural unit with a functionality thatenables reproducibly metering of an aliquot within a microchannelstructure before the aliquot is transported further downstream in amicrochannel structure of a microfluidic device.

Subobjects related to the above-mentioned objects correspond to methodsand uses of the microfluidic devices/structural units for transportingand processing the aliquots of liquids. A particular subobject to thefirst object is a method for reducing evaporation caused by wicking fromthe type of nl-aliquot referred to.

The invention is among others based on the recognition that theappropriate surface tension of a liquid is important for controlling aliquid flow in a microsystem. This in particular applies when dealingwith aliquots in the nano-liter range and/or if the control is exertedwithout mechanical valves and pumps, i.e., by driving the transport ofaliquots through a functional unit of the invention by capillary forceand/or inertia force etc. Typical examples of inertia force aregravitational force and centrifugal force. See also under the heading“Means for driving the liquid flow”.

I. Microfluidic Devices.

The microfluidic device of the present invention typically comprisesone, two, three, four or more sets of microchannel structures in whichaliquots are transported or processed for various purposes, for instanceanalytical or synthetic purposes. The prefix “micro” contemplates thatan individual microchannel structure comprises one or more cavitiesand/or channels that have a depth and/or a width that is ≦10³ μm, suchas ≦10² μm. The lower limit for the width/breadth is typicallysignificantly larger than the size of the largest reagents andconstituents of aliquots that are to pass through a microchannel. Thevolumes of microcavities and thus also of aliquots to be transported andprocessed are typically ≦1000 nl, such as ≦500 nl or ≦100 nl or ≦50 nl.The nl-range comprises, if not otherwise specified, volumes <5000 nl,e.g., within the ranges specified in the preceding sentence. There mayalso be larger cavities, e.g., directly connected to inlet ports, with avolume within intervals, such as 1-10 μl, 1-100 μl, and 1-1000 μl(μl-range). These cavities are typically used for the introduction ofsamples that are to be concentrated within a microchannel structure, orof washing liquids and the like.

The term “microconduit” means a part of a microchannel structure.

A microconduit may be intended for transport of liquids (liquid flowmicroconduits) or for transport into or out of the microchannelstructure (air microconduits). The dimensions of the two types may bedifferent, for instance an air microconduit may have a smallercross-sectional area and/or a higher aspect ratio (depth:width) comparedto a liquid flow microconduit or vice versa. The liquid flowmicroconduit thus may have an aspect ratio ≦1 while an air microconduitmay have an aspect ratio ≧1 or vice versa, or the aspect ratio may beequal for the microconduit irrespective of their use. A liquid flowchannel typically has hydrophilic inner surfaces as discussed elsewherein this specification while an air channel typically has hydrophobicinner surfaces. Liquid flow microconduits may also be used for ventingair into or out of a microchannel structure.

The terms “inlet port” and “outlet port” contemplate port for air andports for liquids.

A microchannel structure may comprise a number of functional units thatare necessary to carry out a predetermined protocol within thestructure. A microchannel structure thus may comprise one, two, three ormore units selected amongst inlet ports, outlet ports, units fordistributing samples, liquids and/or reagents to individual microchannelstructures, microconduits for liquid transport, units for definingliquid volumes, valving units, units venting to ambient atmosphere,units for mixing liquids, units for performing chemical reactions orbioreactions, units for separating soluble constituents or particulatematerials from a liquid phase, waste liquid units including wastecavities and overflow channels, detection units, units for collecting analiquot processed in the structure and to be transferred to anotherdevice e.g., for analysis, branching units for merging or dividing aliquid flow, etc. In one and the same microchannel structure there maybe several inlet ports and/or several outlet ports that are connected toa main flow path via microconduits at a different or at the samedownstream position. These microconduits may also contain functionalunits of the type discussed above.

Typically a microfluidic device comprises in total ≧50, such as ≧100 or≧200, microchannel structures per microfluidic device. The microchannelstructures of a set are essentially identical and may or may not extendin a common plane of a substrate. There may be channels providing liquidcommunication between individual microchannel structures of a set and/orto one or more other sets that may be present in the same device. Themicrochannels are typically covered, i.e., surrounded by walls or othermeans for directing the flow and to lower evaporation. Openings such asin inlet ports, outlet ports, vents etc. are typically present whereappropriate.

The cross-section of a microchannel may have rounded forms all around,i.e., be circular, ellipsoid etc. A microchannel may also have inneredges, i.e., have cross-sections that are triangular, squaric,rectangular, partly rounded, planar etc. Microcavities or microchambersmay have the same or a different cross-sectional geometry compared tosurrounding microconduits.

If not otherwise indicated the term “edge” of a microconduit will referto the intersection of two inner walls of the microconduit. This kind ofedges is typically extending more or less in parallel along theflow-direction (length-going edges). See FIG. 1 that shows amicrochannel having a rectangular cross-section (101), four inner walls(102) with four wall intersections or edges (103). The arrow (105) givesthe flow direction.

A circumferential zone of a microchannel is also illustrated in FIG. 1.It is an inner surface zone (104) in the inner wall of a microchanneland extends in a sleeve-like manner around the flow direction (105). Thelength of this kind of zone is at least from 0.1-10, 0.1-100, 0.1-1000or more times the breadth or depth of the microchannel/microconduit atthe upstream end of the zone.

The microfluidic device may have an axis of symmetry that is n-numbered(C_(n)) where n is an integer between 2 and ∞, preferably 6, 7, 8 andlarger, for instance ∞. In preferred cases the microfluidic device assuch may have a cylindrical, spherical or conical symmetry (C_(∞))and/or is disc-shaped. Axes of symmetry may be combined with utilizingcentrifugal force created by spinning around the axis of symmetry fordriving a liquid flow through a microchannel structure.

The microfluidic device is typically in the form of a disc with themicrochannel structures extending in a plane parallel to the disc plane.

The devices can be manufactured as summarized in WO 2002074438.

The devices are preferably of the same dimension as a conventional CD,but may also be smaller, for instance down to 10% of conventional CDs,or larger, for instance up to more than 200% or more than 400% of aconventional CD. These percentage values refer to the radius.

In the preferred variants the microchannel structures comprises innersurfaces that have been hydrophilized, for instance as described in WO0056808. If necessary the inner surfaces may be coated with a non-ionichydrophilic polymer as described in WO 0056808 or and U.S. Pat. No5,773,488 (Gyros AB), for instance. The preferred variants are the sameas given in these publications, e.g., to a wettability allowing forcapillarity to draw a liquid into a structural unit once having passedthe inlet thereof. Where appropriate hydrophobic surface breaks areintroduced as outlined in WO 9958245 and WO 2002074438. See also WO0185602 (Åmic AB & Gyros AB), which are incorporated herein byreference.

The exact demand on liquid contact angles(hydrophilicity/hydrophobicity) of inner surfaces of the microchannelstructure may vary between different functional units. Except for localhydrophobic surface breaks the liquid contact angel for at least two orthree inner walls of a microconduit at a particular location should bewettable (hydrophilic) for the liquid to be transported, with preferencefor liquid contact angles that are ≦60°, such as ≦50° or ≦40° or ≦30° or≦20°. In the case one or more walls have higher liquid contact angles,for instance non-wettable (hydrophobic), this can be compensated by alowered liquid contact angle on the remaining walls. This may beparticular important if non-wettable lids are used to cover openhydrophilic microchannel structures. The values above apply for theliquid to be transported and to the functional units given above (exceptfor local hydrophobic surface breaks) and at the temperature of use.Surfaces having water contact angles within the limits given above mayoften be used for other aqueous liquids.

The terms “wettable surface” and “hydrophilic surface” are mostlycontemplated a surface that has a liquid contact angle of ≦90° (inparticular for water and other aqueous media). Surfaces that are“non-wettable” or “hydrophobic” thus typically have a liquid contactangle ≧90°. The liquid contact angle in the normal case refers toequilibrium contact angles although it sometimes may refer to recedingand/or advancing contact angles depending on the purpose of ameasurement. In the context of the invention equilibrium contact anglesare primarily contemplated.

A. Valve Functions

Three categories of valves that previously have been suggested formicrofluidic devices are: 1) mechanical valves; 2) valves that compriseintersecting channels together with means that determine through whichchannel a liquid flow shall be created; 3) inner valves, i.e., valves inwhich the passage or non-passage of a liquid depends on physical and/orchemical properties of the liquid and the material in the surface of theinner wall of a microconduit at the position of an inner valve.

Type 1 valves typically require physically closing of a microconduit aretherefore called “closing valves”. They often have movable mechanicalparts for closing a microconduit.

Type 2 valves function without closing and are therefore “non-closing”.A typical example is directing an electrokinetic flow at theintersection of two channels by switching the electrodes. See forinstance U.S. Pat. No. 5,716,825 (Hewlett Packard) and U.S. Pat. No.5,705,813 (Hewlett Packard).

In type 3 valves, the non-passage or passage of a liquid may be basedon: (a) changing the cross-sectional area in a microconduit at the valveposition by changing the energy input to the material of the wall in themicroconduit (closing valves), and/or (b) locally changing theinteraction energy between a through-flowing aliquot and an innersurface of a microconduit at the valve position (non-closing valves),and/or (c) a suitable curvature of the microconduit at the valvefunction (geometric valves, non-closing).

Type 3a valves are illustrated in WO 0102737 (Gyros AB) in whichstimulus-responsive polymers (intelligent polymers) are suggested tocreate a valve function, and in WO 9721090 in which relaxation ofnon-equilibrium polymeric structures and meltable wax plugs aresuggested to create a valve function.

In type 3b valves, the microconduit at the position of the valve is openeven if the liquid is stopped (inner valves including capillary valves,also called passive valves). Through flow in this kind of valves isaccomplished simply by increasing the force driving the liquid. The useof hydrophobic surface breaks (changes in chemical surfacecharacteristics) as valves is described in for instance WO 9958245, WO0146465, WO 0185602 (Amic AB & Gyros AB), WO 0187486 and WO 2002074438.The use of changes in geometric surface characteristics as valves isdescribed in for instance WO 9615576 (David Sarnoff Res. Inst.), EP305210 (Biotrack), and WO 9807019. Type 3b valves comprise ananti-wicking function if they utilize changes in chemical and/orgeometrical surface characteristics in edges as described foranti-wicking means.

Type 3c valves may be achieved by linking an upward bent of amicrochannel immediately downstream to a downward bent in centrifugalbased systems. This is illustrated in WO 0146465 that suggestsconnecting an upward bent microconduit downstream to a U/Y-shapedmicroconduit.

B. Anti-Wicking Means

Anti-wicking means are typically local surface modifications thatcounteract wicking/imbibing.

Imbibing (wicking) means that liquid transport is initiated in the edgesof micro channels. See for instance Dong et al (J. Coll. InterfaceScience 172 (1995) 278-288) and Kim et al (J. Phys. Chem. B 101 (1997)855-863). Imbibing renders it difficult to retain a defined volume of aliquid in a desired microcavity for a longer period of time in casethere is a microconduit having a length-going edge directly connected tothe microcavity. This in particular applies if the volume≦5 μl, such asin the nl-range or less. If the microconduit is connected to ambientatmosphere, for instance via an inlet port, imbibing will promoteevaporation and irreversible loss of a predispensed volume of a liquid.

Anti-wicking means typically comprises a change in surfacecharacteristics, such as in geometric and/or chemical surfacecharacteristics, in an inner edge of a microconduit. The edge typicallystarts in a microcavity and stretches into the microconduit.Anti-wicking means may be present upstream or downstream a microcavityintended to contain a liquid. An anti-wicking functionality mayinherently also be present in inner valves that are based on thepresence of a hydrophobic surface break in an inner edge.

The change in geometric surface characteristics is typical local and maybe selected from indentations, protrusions (projections), and anincrease in the angle between the two inner walls defining alength-going inner edge. In most cases the deformation will also stretchinto and/or across a wall delineated by this kind of edge, for instanceinto and/or across a wall delineated by two edges comprising thedeformation. For indentations and protrusions this will meanvalleys/grooves and ridges, respectively, across the wall. An increasein the angle between two intersecting walls means in its extreme thatthe inner edge can be rounded within a zone carrying the anti-wickingmeans but not rounded between this zone and the microcavity. Themicroconduit thus may locally be cylindrical. Also other physicaldeformations of the edges may result in anti-wicking.

Deformations in the form of indentations, for instance, may be“ear-like” as illustrated in the figures (214, 406, 509) of the presentspecification or similar to a triangular groove as illustrated in FIG.13 (1312) of WO 2002074438, which is incorporated by reference.

A change in chemical surface characteristics (surface break) in thecontext of anti-wicking primarily refers to a change inhydrophobicity/hydrophilicity of the surface of an inner wall of amicrochannel structure. Typically the inner surface of the microconduitis hydrophilic as discussed above with a change into hydrophobicitywhere anti-wicking is to be achieved.

In a similar manner as for changes in geometric surface characteristicsa change in chemical surface characteristics typically may extend intoand/or across the inner surface of a wall in a microconduit.

A change in geometric and chemical surface characteristics may fully orpartially coincide in the inner surface of microconduit. An indentationand the like which stretches across an inner wall thus physically shouldcomprise the change in chemical surface characteristics if the aim is toavoid valving effects. Compare FIG. 4 (406 and 407).

The anti-wicking means in a circumferential zone that comprise inneredges should be at different positions (or be lacking) in at least onecompared to the position of the anti-wicking means in the other edges ofthe circumferential zone. For instance, if the microconduit has afour-edged cross-section (rectangular) with all four edges extendinginto a microcavity, opposite inner walls typically may have the changein surface characteristics at different distances, e.g., pair-wise, fromthe microcavity. Compare for instance FIG. 4.

The anti-wicking means described herein is adapted to prevent wickingfor aliquots that have a surface tension, which is ≧5 mN/m, such as ≧10mN/m or ≧20 mN/m. The importance of including anti-wicking means isprimarily related to handling of aliquots ≦5 μl, such as aliquots in thenl-range, in the microfluidic devices described herein.

Further information about various kinds of anti-wicking means possiblycombined with an inner valve function is given in WO 2002074438.

C. Means for Driving the Liquid Flow

The liquid flow may be driven in the microfluidic device of the presentinvention by distinct means that either is present on a substratecomprising the microchannel structures or is external to the substrate.The former variants typically means liquid flow created byelectroendosmosis, by micropumps that are present on the substrate,expanding gas etc. The latter variants typically mean externalpressure-generating means that create a liquid flow that is in fluidcommunication with the microchannel structure. Another alternative is touse forces such as capillary forces and inertia force includinggravitational force and centrifugal force. In this latter case no meansfor moving the liquids is required in the microchannel structures or inthe substrates carrying the microchannel structures.

Variants in which the microchannel structures are oriented from an innerposition to an outer position in relation to a spinning axis, such as anaxis of symmetry of a substrate as described above are typicallycombined with a spinner that is capable of spinning the substrate aroundthe spinning axis that may coincide with the axis of symmetry. Usefulspinners should be able to create the necessary centrifugal force fordriving the liquids through at least a part of a microchannel structure.The centrifugal force may be utilized in combination with a secondliquid aliquot to create a sufficient local hydrostatic pressure withina structure to drive a first aliquot through an outward (downward)and/or an inward (upward) bent of a microchannel structure. See forinstance WO 0146465. Typically spinning speeds are within the interval50-25000 rpm, such as 50-15000 rpm. The spinning speed within a givenprotocol may vary and depends on the part structure that is to be passedby a liquid, for instance. In case the microfluidic device contains aplurality of microchannel structures that are to be run in parallel, itmay be beneficial to start the passage of liquid through a particularstructural unit with a short pulse of increased spinning followed by aslower spinning.

D. Orientations and Positions in a Microfluidic Device

The present invention is primarily intended for geometric arrangementsin which the microchannel structure is present in a substrate andarranged about an axis of symmetry (spinning axis) that typically isgoing through substrate. The term “radial distance” means the shortestdistance between an object and the axis of symmetry and/or a spinningaxis The radial distance for an inlet port and a structural unit may bethe same, or the inlet port may be at a shorter or longer radialdistance compared to the structural unit. In a typical case there isalso an outlet port for liquid downstream the structural unit, which inmost cases is at a larger radial distance than the inlet port. Themicrochannel structure may or may not be oriented in a planeperpendicular to the axis of symmetry. The terms “higher” and “upper”for a level/position means that an object is at a shorter radialdistance (inner position) compared to being at a “lower” level/position(outer position). Similarly, the terms “up”, “upward”, “inwards”, and“down”, “downwards”, “outwards” etc. will mean towards and from,respectively, the spinning axis. This terminology applies if nototherwise is specified. With respect to other arrangements/substratesand conventional driving forces, i.e., gravity force, externally appliedpressure, electro-osmotically (electrokinetically, by electroendoosmosisetc.) driven flows etc., these terms have their conventional meaning.

The terms “downstream” and “upstream” are related to the processprotocols and liquid flow as such. The terms thus refer to the order inwhich a unit, a part, a process step, etc. is utilized. A downstreamposition is coming after an upstream position.

II. Structural Units A-E

Inlet ports typically have hydrophobized areas to direct applied liquidinto the ports. Local surface breaks that are hydrophobic for aqueousliquids are represented by straight or bent rectangles. They areprimarily present for controlling liquid flow, e.g., in valves (innervalves), in anti-wicking means, in vents and for directing liquidinwards the structures in inlet ports.

A. Unit A: Retaining Microcavity Unit

The first aspect of the invention is a microfluidic device comprising amicrochannel structure in which there is a structural unit accomplishingretaining of a nl-aliquot of liquid in a microcavity (retainingmicrocavity) as discussed in the first object. The nl-aliquot has beenobtained by mixing two liquid aliquots within the microchannel structureand is henceforth named “mixed nl-aliquot” or “mixed aliquot”.

The present inventors have recognized that a nl-aliquot placed in amicrocavity of a microchannel structure under static non-flow conditionsis quickly reduced in volume and may disappear from the microcavity inthe case the microchannel structure is openly connected to other partsof the structure, e.g., to inlet ports or outlet ports which directlycommunicate with ambient atmosphere and to other microcavities notcontaining liquid. The present inventors have discovered that thiseffect is related to wicking in inner edges, and that the effect isenhanced if evaporation of a wicked liquid from the outlet and/or inletports is possible. The present inventors hereby present a solution tothis problem. The solution for a mixed nl-aliquot is to a) placeanti-wicking means in the microconduits directly connected to amicrocavity intended to retain a well-defined aliquot of liquid, and/orb) secure that the distance within the microchannel structure betweenthe microcavity and each of the outlet ports has a sufficient length.

The structural unit of the first aspect of the invention is illustratedin FIGS. 2 a-b and d-e. The unit is characterized in comprising (a) amicrocavity (retaining microcavity) (219) which is intended forretaining a nl-liquid aliquot (mixed aliquot) under static non-flowconditions and is located between at least one (205,215,237) of said oneor more inlet ports (205,215,237) and at least one (238,241) of said oneor more outlet ports (207,238,239,240,241); (b) a mixing unit(302+303+30i) which is located upstream the retaining microcavity (219)and downstream said at least one inlet port (205,215,237) and in whichtwo or more aliquots (aliquot 1, aliquot 2 etc.) are to be mixed to formsaid mixed aliquot; and (c) two or more microconduits (218,220,242)directly connected to the retaining microcavity (219) and communicatingwith one of said inlet or outlet ports (205,207,215,237,238,239,240,241).

Each of the microconduits (218,220,242) comprises anti-wicking means(221 a,e) in association with the joint between the retainingmicrocavity (219) and the microconduit. Alternatively, if a microconduitis directly attached to a retaining microcavity (219) and does notcontain anti-wicking means, then the distance (d₁) from the retainingmicrocavity (219) to an opening to ambient atmosphere in an inlet oroutlet port (205,207,215,237, 238,239,240,241) is ≧10 times the largestcross-sectional dimension of this microconduit at its joint to theretaining microcavity (219).

The distance (d₁) refers to the shortest distance, if alternatives areavailable. The distance is measured inside the microchannel structureand includes length of the microconduit concerned. The cross-sectionaldimension refers to an inner dimension.

The effect of reducing evaporation to ambient atmosphere withoutanti-wicking means may be further enhanced if the distance (d₁) isfurther increased, e.g., to ≧20 times or ≧50 times or ≧100 times or ≧500times or ≧1000 times ≧5000 times the largest cross-sectional dimensionof this microconduit at its joint to the retaining microcavity (219).

In principle the above-mentioned conditions for microconduits notcontaining anti-wicking means may be applied to microconduits containinganti-wicking means. Accordingly any microconduit connected to aretaining microcavity (219) may contain both anti-wicking means andcomply with the conditions for the distance (d₁).

The microconduits connected to the retaining microcavity may be eitheran air microconduit (242, FIG. 2 d) or a liquid flow microconduit(218,220, FIGS. 2 a-b and c-d). The latter typically also functions as amicroconduit for venting out air displaced by an incoming liquid.Further differences between the two kinds of microconduits are discussedunder the heading “Microfluidic device”.

One, two or more up to all of the microconduits (218,220,242) directlyconnected to the retaining microcavity (219) have one or morelength-going edges extending continuously from said retainingmicrocavity. Each of these edges preferably has anti-wicking means. Airmicroconduits having hydrophobic inner surfaces at their joint to theretaining microcavity (219) will inherently provide anti-wicking means.See further under the heading “Anti-wicking means”.

A liquid flow microconduit (218,220) directly connected to a retainingmicrocavity (219) typically also comprises a non-closing valve functionin association with the joint between the microconduit (218,220) and themicrocavity (219). This valve function may be based on a change ingeometric and/or chemical surface characteristics and/or on a suitablecurvature of these flow microconduits (upward bents) as illustrated inFIGS. 2 a-b and e (microconduit 220). The anti-wicking means and thevalve functions may fully or partially coincide in a liquid flowmicroconduit (218,220). See further under the heading “Valve functions”.

The mixing unit of the microchannel structure may in principle be anykind of mixing unit that can be adapted to the instant kind ofmicrofluidic structures. This includes the kind of mixing unitsdiscussed under the heading “Background publications mixing units (unitB)” and the innovative unit B discussed below. Thus the mixing unit maycomprise two inlet microconduits (224 and 225) for the aliquots to bemixed (aliquot 1, aliquot 2). These inlet microconduits merge in thedownstream direction into a common microconduit (302) that communicateswith the retaining microcavity (219) in the downstream direction. At theintersection of the two inlet microconduits (224 and 225), there may bea microcavity (303) with a total volume that is essentially the same asor larger than the total volume of the aliquots to be mixed andintroduced via the inlet microconduits. There may also be further inletmicroconduits merging at the intersection or elsewhere for mixing ofadditional aliquots with aliquot 1 and aliquot 2. Mixing may occur inthe common microconduit (302) (mixing microconduit), or in themicrocavity (303) (mixing microcavity). The preferred mixing unit isaccording to unit B below.

As discussed above a plurality of microchannel structures may bearranged around a spinning axis combined with using centrifugal forcecreated by spinning around the spinning axis for driving the liquid flowin parallel through the structures. Centrifugal force may be combinedwith capillary force. Other forces may also be used for this and otherconfiguration. See for instance under the heading “Means for driving theliquid flow”.

In particular a plurality of the microchannel structures may be presenton a microfluidic device that has an axis of symmetry coinciding with aspinning axis. In this variant the microchannel structures are typicallyarranged to permit the use of centrifugal force for driving a liquidflow in parallel within individual microchannel structures. See aboveunder the heading “Microfluidic device”.

The retaining microcavity (219) may have different forms as known in thefield. Preferred variants often define or are part of a U/Y-shapedstructures, possibly linked to upwardly bent microconduits at either oneor both of the upwardly directed shanks of the U/Y as describedpreviously for reaction microcavities (WO 0040750, WO 0146465). TheU-shaped structure may also be as presented in FIG. 2 e where the U isdefined by a reaction microcavity which comprises two upwardly directedshanks, the upper parts of which are connected to microconduits(218,220) containing the anti-wicking means/valves (221 e,a).Microconduit (218) plus the most downstream part of the retainingmicrocavity (219) define an upward bent that will provide a valvefunction. This latter variant may be advantageous in the case the mixedaliquot is to be transported further downstream in the structure. SeeFIG. 2 a-b and e. Another variant is that the microcavity (219) iscircle like with the down stream or upstream microconduit attachedwithout formation of this kind of bent. See for instance FIG. 2 e inwhich one of the microconduits (242) is a pure air channel whichpreferably has an hydrophobic inner surface that in fact creates ananti-wicking effects and renders passage of liquids difficult.

The mixed aliquot may be retained in the microcavity (219) for differentpurposes, such as performing a chemical and/or biochemical reactionand/or a measurement of one or more chemical or physical parameters ofthe mixed aliquot under static non-flow conditions within themicrocavity (219) with a high accuracy (that would have suffered fromloss of liquid and changes in concentrations if wicking and evaporatingwould have been allowed to act). Typically the reaction and/ormeasurement are part of an assay procedure for determining/detecting acomponent present in the mixed aliquot or in some other aliquotsdispensed to the microchannel structure. The reaction may also beperformed for synthetic purposes. Biochemical reactions includebioaffinity reactions (e.g., reactions between an antibody and anantigen/hapten, an enzyme and its substrate, cofactor, cosubstrate etc.,complementary nucleic acids, and lectin carbohydrate) including enzymereactions, cell reactions, etc. The reactions may take place in ahomogeneous liquid phase or involve reactions between solid phase boundreactants and dissolved reactants or reactants in suspended form(heterogeneous reactions). Retaining may also be for the storing of themixed aliquot, for instance awaiting certain process steps to take placeoutside or inside the microfluidic device on other aliquots that are tobe used in the microfluidic device, possibly together with the mixedaliquot. The periods of time for retaining are as outlined in the firstobject. After the retaining period has lapsed, further processing of theliquid aliquot may take place in the reaction microcavity (219) orfurther downstream in the microchannel structure.

The surface tension of the liquid, the liquid contact angles of theinner surfaces of the microchannel structures, kind of liquids etc. areselected as described under the headings “Microfluidic device”, “Valvefunctions” and “Anti-wicking means”.

The use of unit A is defined by the method of the second main aspect ofthe invention and comprises in addition a mixing step utilizing themixing subunit and a process step of the mixed aliquot that may beperformed for any of the reasons discussed above.

B. Unit B: Mixing Unit

The second subaspect of the invention is a microfluidic device asdefined herein comprising a microchannel structure in which there is astructural unit accomplishing mixing of aliquots (unit B).

This subaspect is based on our recognition that quick, efficient andreliable mixing of aliquots that are miscible can take place by firstcollecting the aliquots in a microcavity, preferably under the formationof a phase system, and then permitting the aliquots to pass through amicrochannel of sufficient length to permit homogeneous mixing.

Preferred variants of our mixing units are illustrated in FIGS. 2 a-c.The variants shown are arranged as discussed above on a spinnablesubstrate (compare the arc-like arrangement). FIGS. 2 a-b comprises fourmicrochannel structures connected to each other by a common distributionchannel.

In general terms unit B comprises an inlet arrangement (201) and amixing microconduit (202) as described in prior publications. Betweenthe inlet arrangement (201) and the mixing microconduit (202) we haveintroduced a microcavity (203) to precollect the aliquots to be mixed inthe mixing microconduit (202). The precollecting microcavity (203) hasan opening (223) in its lower part which opening is in register with themixing microconduit (202). This precollecting microcavity may havevarious designs with one feature being that it should enable formationof a liquid interface between the two aliquots to be mixed. The flowdirection should be essentially perpendicular at the interface, i.e.,90°±45°.

In addition to the mixing unit as such, FIGS. 2 a-b show:

(a) A common distribution channel (204) as described for unit C belowwith an inlet port (205) with ridges/projections (206,216) as describedfor unit D above, an outlet port (207), and inlet vents (208) to ambientatmosphere via a common venting channel (209) and an air inlet (237).When the distribution channel is filled with liquid and a downwarddriving force is applied, liquid will be forced out through themicroconduits connecting the distribution channel (204) with themicrocavities (203). At the same time air will enter through the vents(208);

(b) A common waste channel (210) comprising outlet ports (238);

(c) Volume-defining units (213) as described for unit E and comprisinganti-wicking means (214,221 g) as described above, an inlet port (215)with ridges/projections (206,216) as described for unit D, and anoverflow channel (217) ending in an outlet (212) in the common wastechannel (210); and

(d) A microcavity (219) in which various kinds of processes may becarried out as discussed elsewhere in this specification, and anenlarged waste outlet conduit (220), which merges into the common wastechannel (210) via the outlet (211).

Surface breaks (non-wettable) are represented by straight or arc-formedrectangles (e.g., 221 a,b,c etc. and 222, respectively).

The mixing unit of the present invention is characterized by comprising(a) the microcavity (203) with an outlet opening (223), typically in itslower part; (b) an inlet arrangement (201) linked to the microcavity(203), and (c) a mixing microconduit (202) connected to the outletopening (223). The microcavity (203) shall have a volume sufficient tocontain simultaneously the aliquots to be mixed. The inlet arrangementis connected to the upper or lower part of the microcavity (203).

Preferably there is a valve associated with the mixing conduit (202),preferably close to its joint to microcavity (203). This valve functionis preferably an inner valve of the same kind as discussed elsewhere inthis specification, for instance in the form of a surface break(non-wettable) (221 b). The valve may also be mechanical.

The inlet arrangement may comprise a common inlet microconduit (notshown) for several aliquots and/or separate inlet microconduits (224 and225) for individual aliquots. The joint between these microconduits andthe inlet openings are preferably located at the upper part ofprecollecting microcavity (203). In the upstream direction each of theseinlet microconduits (224 and 225) communicates with an inlet port (205and 215). Each inlet microconduit (224 and 225) may comprise asubmicrocavity permitting separate predispensing of an aliquot to amicrochannel structure before transport down into the microcavity (203).In FIGS. 2 a-b one of these submicrocavities is microcavity (226) of thevolume-defining unit (213) and the other Y-shaped structure (227) a partof which belongs to the common distribution channel (204). Between eachsubmicrocavity (226,227) and microcavity (203) there may be a valvefunction (221 d,c, respectively) that enables for aliquots to betransported into the submicrocavities (226,227) without leakage into themicrocavity (203). The valve function at these positions is preferablyan inner valve of the same kind as discussed for the valve functions(221 a,b) associated with the mixing microconduit (202), e.g., a surfacebreak (non-wettable) (221 a,b).

As illustrated in FIGS. 2 a-b the mixing conduit (202) may have variousforms. It may be a single channel that is meandering or coiled in orderto save space as suggested in FIG. 2 a. It may also be built up of achain of interlinked small microcavities (228), each of which has asmoothly increasing cross-sectional area from the inlet end and asmoothly decreasing cross-sectional area when approaching the outlet endas suggested in FIG. 2 b. FIG. 2 b also illustrates that these smallmicrocavities can be of continuously increased breadth from their inletand outlet ends with the steepest increase from the outlet end(droplet-shaped breadth).

When the aliquots are introduced into microcavity (203) there should beformed a phase system in the microcavity. Each aliquot should berepresented by a liquid phase. The flow direction out of the microcavity(203) should be essentially perpendicular to the interface between thephases. During passage of the phase system into the mixing microconduit(202), the upper phase is typically entering in the center of themicroconduit and the lower phase next to the inner wall. Mixing willoccur during the transport in the microconduit (202) probably due to thefact that the center of the liquid flow will have a higher flow ratethan the peripheral part next to the inner wall of the mixing conduit.This means that the two aliquots repeatedly will replace each other inthe front position while traveling through the mixing microconduit. Thismay be the reason for the quick and efficient mixing that isaccomplished in the inventive mixing structure. If the mixingmicroconduit (202) is of sufficient length in relation to the flow rateand the constituents of the aliquots, complete mixing will have occurredat the end of the mixing microconduit (202). Sufficient length typicallymeans that the phase system should have a smaller volume than the volumeof the mixing microconduit (202).

FIG. 2 c shows a third variant of the inventive mixing unit. Thisvariant has a microcavity (229) corresponding to microcavity (203) inFIGS. 2 a-b. The microcavity (229) comprises an upper downward bent(230) and a lower downward bent (231) and a channel part (232) goingfrom the lower part of the upper bent (230) to the lower part of thelower bent (231). In the lower part of the lower bent (231) there is anopening (233) leading into a mixing microconduit (234). Preferably thereis a valve (235) in the mixing microconduit (234), typically close tothe opening (233). This valve preferably is an inner valve for instancecomprising a change in surface characteristics (non-wettable surfacebreak). FIG. 2 c in addition shows inlet vents to ambient atmosphere(236 a-d) at top positions of the bents. When filling the downward bentswith aliquot 1 and aliquot 2, respectively, a liquid interface can beformed in the communicating microconduit (232). By applying a downwardlydirected driving force the two aliquots will be forced into the mixingmicroconduit in the same manner as for the variants described in FIGS. 2a-b.

In the variant of FIG. 2 c, the inlet arrangement of FIGS. 2 a-b isfully integrated with the precollecting microcavity (203) and thereforemore or less indistinguishable.

The microcavity (229) of FIG. 2 c may be part of two aligned commondistribution channels of the same kind as outlined in FIGS. 2 a-b.

In preferred variants, a microchannel structure comprising unit B may beoriented about a spinning axis that in turn may coincide with an axis ofsymmetry of a spinnable substrate/device as discussed elsewhere in thisspecification. The flow direction through the outlet opening ofmicrocavity (203) is typically oriented essentially outward in relationto the axis of symmetry (spinning axis).

The use of unit B comprises a method for mixing two or more aliquotswithin a microfluidic device comprising a microchannel structure. Thealiquots may have the same or different volumes and/or compositions. Themethod is characterized in comprising the steps of: (i) providing amicrochannel structure comprising unit B as defined above; (ii)introducing the aliquots via the inlet arrangement of unit B intomicrocavity (203), preferably to form a phase system therein; (iii)applying a driving force to transport the phase system through mixingmicroconduit (202); (iv) collecting the homogeneously mixed aliquots atthe end of the mixing microconduit (202) for further transport and/ortreatment within the microchannel structure.

If submicrocavities (226,222) are present in the inlet arrangement(201), the aliquots to be mixed may be individually predispensed tothese submicrocavities before the driving force for transport intoprecollecting microcavity (203) is applied.

The rules for selecting driving force are the same as discussed asdiscussed above. For spinnable substrate centrifugal force is preferred.

At least one of the aliquots should have a surface tension, which is ≧5mN/m, such as ≧10 mN/m or ≧20 mN/m.

Common waste channel: In FIGS. 2 a-b the common waste channel (210) havesupporting means for minimizing the risk for collapse due to the breadthof the channel. The surface break (227) improves the emptying of theoverflow channel (217) and facilitate its refilling.

C. Unit C: Unit for Forming a Plurality of Aliquots of Defined Volumeswithin a Microfluidic Device, Distribution Manifold

The third subaspect of the invention is a microfluidic device comprisinga microchannel structure in which there is a structural unit (unit C)accomplishing metering one, two, three or more aliquots (two ormore=plurality of aliquots).

This subaspect is based on our recognition that the relative loss ofliquid by evaporation may be significant when dispensing small aliquots,in particular nl-volumes, to individual microchannel structures in amicrofluidic device. We have also found that the prior systems utilizinga common reagent fill channel from which metering is done in parallel ina plurality of metering microcavities are insufficient when thecross-sectional dimensions of the channels are in the lower part of theμm-range and/or the volumes are decreased into the nl-range.

Unit C presents a solution to these problems and makes it possible toreproducibly meter a number of smaller aliquots within the samemicrofluidic device and to transport these aliquots in parallel intoseparate microchannel structures of the microfluidic device or intoseparate parts of the same microchannel structure. The aliquots may beidentical or different with respect to size, composition etc., and aretypically in the nl-range as defined above

Unit C is represented in FIGS. 3 a-c that show variants that arearranged about a spinning axis that may coincide with an axis ofsymmetry as discussed above. In these figures the distribution unit assuch is encircled (300).

Based on FIGS. 3 a-b, the unit comprises: (a) a continuous microconduit(301) containing an upper part at each end (end parts, 302, 303) andtherebetween alternating lower and upper parts (304 a-h/f and 305 a-e,respectively); (b) the number of upper parts including the end parts isn and the number of lower parts is n-1 where n is an integer >2, i.e.,≧3; (c) each of the upper parts (302, 303, 305 a-e/g) has means forventing (top vent, inlet vents) (306 a-g/i) to ambient atmosphere and/oranti-wicking means (326 a-i) in length-going edges delineating its lowerwall(s); (d) each of the lower parts (304 a-f/h) has an emptying openingwhich in a downstream direction via a connecting microconduit (307a-f/h) communicates with a substructure of a microchannel structureand/or with a corresponding substructure of another microchannelstructure; (e) each of the connecting microconduits (307 a-f/h) has avalve (308 a-f/h), i.e., a valve function in close association with thejoint between the connecting microconduit and the corresponding lowerpart; (f) an inlet port (309) is connected to the continuousmicroconduit (301) directly or indirectly at one of the upper parts(302, 303, 305 a-e/g), preferably via one of the end parts (302 or 303);and (g) an outlet port (310) is connected to the continuous microconduit(301) directly or indirectly at another upper part (302, 303, 305a-e/g), preferably via one of the end parts (302 or 303) (whichpreferably is not connected to the inlet port, i.e., an inlet port andan outlet port should not connected at the same upper part).

In a lower part (304 a-f/h), the continuous microconduit (301) ispreferably shaped as a downward bent. This kind of bents includes thatthe microconduit in the bent is enlarged to a microcavity. Similarly anupper part is preferably in the form of an upwardly bent microconduitbut without enlargement of the type that can be present in a downwardbent.

The smallest cross-sectional areas of the continuous microconduit (301)between the ends (302, 303) should be in the upper parts, withpreference for in association with the top vents (306 a-g/i) and/or theanti-wicking means (326 a-i). The cross-sectional area of the continuousmicroconduit (301) may be of constant size and/or shape along the lengthof the continuous microconduit.

The inlet ports (309) and the outlet ports (310) are typically at alower level than the extremes of the upward bents and may even be at alower level than the extremes of the lower parts (304) and/or than adesired part of the individual microchannel structures that aredownstream the lower parts (304) (for instance at a lower level than awaste outlet port).

The liquid aliquot is preferably transported from an inlet port (309) toan outlet port (310) of the continuous microconduit (301) by capillaritymeaning that the liquid contact angle in this part of the microchannelstructure continuously has to be well below 90°, i.e., preferably ≦40°,such as ≦30° or ≦20°, and enabling filling by capillarity of thecontinuous microconduit (301) to valves (308 a-f/h) by self-suction froman inlet port of the microconduit (301).

In the preferred variants the continuous microconduit (301) hasmeander-form.

The integer n is preferably ≧2, such as 3, 4, 5, 7, 8, 9, 10, 11, 12 ormore.

The joints between a connecting microconduit (307 a-f/h) and a lowerpart (304 a-f/h) are preferably located at the same level and/or at thelowest part of a downward bent. The valves (308 a-f/h) in the connectingmicroconduit (307 a-f/h) preferably are inner valves that may be closingor non-closing.

The top vents (306 a-g/i) are preferably located at the same level onthe upward bents (302, 303, 305 a-e/g). Each top vent (306 a-g/i)comprises an opening in an upper part (302, 303, 305 a-e/g) of thecontinuous microconduit (301) and possibly also a microconduit. Each topvent may have an inner valve and/or may be equipped with anti-wickingmeans in the case the top vent has a length-going edge that mightpromote imbibing and evaporation of liquid. The anti-wicking means aredescribed elsewhere in this specification. The top vents may beconnected via a common venting channel (311) and an inlet (325) toambient atmosphere.

The openings associated with top vents in the upper part may be directedupward as illustrated in FIGS. 3 a-c but may also be directed in otherdirections, e.g., as illustrated in FIG. 2 c (236 a-d).

As outlined for unit D preferred variants of unit C may have: A) inletport (309) designed with a hydrophobic surface break at the rim of theinlet opening which directs a dispensed aliquot into the opening of theport, and B) an inner valve in the microconduit connecting an upper partto an inlet port (310). Compare also FIG. 7 and FIG. 8 of WO 2002075775and WO 2002075776, respectively, which are incorporated herein byreference.

One or both of the end parts (302,303) may directly or indirectly beconnected to another distributing unit C according to the invention asillustrated in FIG. 2 of WO 2002075312, which is incorporated herein byreference.

Unit C is intended for distributing (n-1) aliquots to (n-1) microchannelstructures or (n-1) part structures of a microchannel structure. Thevolume between two close top vents (306 a-e/g) will in most variantsdefine the volume of the aliquot to be dispensed through the connectingmicroconduit (307 a-f/h) between these top vents (segment). By varyingthe depth and/or width of different segments, one can envisage that thevolumes dispensed through different connecting microconduits (307 a-f/h)can differ in a controlled manner.

By first filling the continuous microconduit (301) with liquid betweenits end parts (302 and 303), for instance by self-suction, and thenforcing liquid to pass through the connecting microconduits (307), themetered aliquots between close top vents will pass into separateconnecting microconduits. Spillover between neighboring segments of thecontinuous microchannel (301) will be minimized due to the top ventsand/or by the presence of anti-wicking means (326) in edges delineatinglower walls in upper parts.

By filling the segments with the same liquid, for instance in one step,aliquots of the same composition will be dispensed through all theemptying openings.

FIG. 3 b illustrates a non-meander form of unit C (straight form) inwhich the lower parts (304 a-h) are in form of microcavities that areconnected to each other via upper parts (305 a-g). At the end of thecontinuous microconduit (301) there are also upper parts (302,303) viawhich an inlet and an outlet port may be connected (309 and 310,respectively). Means for venting (306 a-i) the continuous microconduit(301) may be associated with upper parts of the continuous microconduit,for instance in the conduit parts (305 a-g) and/or in the end parts(302,303). The lower part of each microcavity (304 a-h) has an outletopening to which a connecting microconduit (307 a-h) with a valvefunction (308 a-h) is associated. There may also be anti-wicking means(rectangles, 326 a-i) at both sides of each microcavity (304 a-h) inedges that extend down into a neighboring microcavity/lower part (304a-h). The anti-wicking means may be of the same kind as discussedelsewhere in this specification. A variant is shown in FIG. 7 and FIG. 8of WO 2002075775 and WO 2002075776, respectively, which are incorporatedherein by reference, and illustrates a distribution manifold with acentrally located inlet port and anti-wicking means in the edges asdiscussed above but without the top vents (306 a-g).

FIG. 3 c represents a variant, which will enable distribution ofaliquots of different compositions to individual microchannelsubstructures. The distribution unit as such is encircled (300).Upstream the distribution unit (300) there is a microchannelsubstructure (311), which will enable filling of segments between closetop vents (306 a-d) of the continuous microchannel (301) with aliquotsof different compositions. In order to accomplish this, substructure(311) comprises a volume-defining unit (312), which is capable ofmetering a liquid volume that is equal to the volume of the segmentbetween two close top vents (306 a-d) in the continuous microchannel(301). If the volumes of the segments are different, subunits definingdifferent volumes may be included in substructure (311). In FIG. 3 c,the substructure (311) upstream the distribution unit (300) may comprisefurther functionalities. Thus substructure (311) may comprise a firstdownward bent (313) which has one of its shanks (314) connected to theend part (302) of the continuous microchannel (301) and the other shank(315) connected to the lower part of a second downward bent (316) thatin turn is connected to a metering part of volume-defining unit (312) atthe upper part of one of its shanks (317). The other shank (318) of thesecond downward bent (316) may be venting to ambient atmosphere via aninlet (327). The illustrated metering part of volume-defining unit (312)is of the same kind as unit E and includes an overflow system and aninlet port (319) of the same kind as unit D. The volume of the meteringmicrocavity (320) of the volume-defining unit (312) is the same as in asegment between two close top vents (306 a-d). The substructure (311) ofFIG. 3 c also comprises (a) a large waste chamber (321) with arelatively wide opening (322) into the lowest part of the first downwardbent (313), and (b) a valve function (323) associated with theconnection between the first and second downward bent (313,316).

Due to the size of the waste chamber (321) there are supporting means inform of pillars (324) securing that its top and bottom are kept apartfrom each other.

The kind of design presented in FIG. 3 c makes it possible toconsecutively fill the segments between the top vents (306 a-d) of thecontinuous microconduit (301) with aliquots of different compositions,and thus to distribute aliquots of different composition to theindividual substructures connected to unit C via the connectingmicroconduits (308 a-c ). With reference to FIG. 3 c this means(presuming waste chamber (321) is closed or absent):

Step 1: Aliquot 1 is metered in the volume-defining unit (312) andtransported to downward bent (313), for instance by spinning if the unitis present on a spinnable substrate that may be a circular disc by thehydrostatic pressure created by centrifugal force.

Step 2: Aliquot 2 is metered in the volume-defining unit (312) andtransported into the downward bent (313). This will push aliquot 1 tosegment 1 (between top vents 406 a and b) of the continuous microconduit(301).

Step 3: Aliquot 3 is metered in the volume-defining unit (312) andtransported into the downward bent (313). This will push aliquot 1 tothe second (next) segment and place aliquot 2 in the first segment.

When the desired number of segments has been filled a downwardlydirected driving force is applied to pass the aliquots through theirrespective connecting microconduit/valve (307 a-c /308 a-c ).

A simplified variant of FIG. 3 c means that the first downward bent(313) is designed as a volume-defining unit, for instance by placing anoverflow system at the same level as the top vents (306 a-d) of thecontinuous microconduit (301) in shank (315).

By introducing a chemical functionality, for instance in the form ofsubstructure comprising an inlet port followed by a reaction zone infront of unit C, unit C may be used for collecting separate fractionsbetween each pair of neighboring top vents in the continuousmicroconduit (301) from liquids that have passed through the reactionzone. Collected fractions can then be further processed, for instanceanalyzed, by taking them further down into the microchannel structurevia the connecting microconduits (307 a-c). With respect to FIG. 3 c,such a zone suitably is positioned between the first and second downwardbents (316 and 313, respectively), for instance combined with the valve(323).

The reaction zone may for instance comprise an immobilized reactantselected from (a) a catalysts such as an enzyme, (b) a ligand capable ofbinding to a component of a liquid which is to pass through the zone,(c) an affinity complex between a ligand and a binder etc. Based on thepresence of particular components in the fractions that are collectedone can analyze for features related to the zone as such or to theliquids applied, e.g., features of compounds present in the zone and/orin a fraction.

Unit C is preferably present in a spinnable microfluidic device of thekind discussed elsewhere in this specification. The continuousmicroconduit (301) may then be oriented in an annular-like fashionaround a spinning axis and may occupy at least a sector of an annularzone defined by the continuous microconduit. The sector typically coversat least 0.5-10° and at most 360° relative a spinning axis and/or anaxis of symmetry. The lower parts (304) of the unit are directedoutwards from the spinning axis and the upper parts (302, 303, 305)inwards towards the spinning axis.

The driving force is selected according to the same principles asoutlined for the microfluidic device above, with preference capillarityfor filling the continuous microconduit (301) and centrifugal force orovercoming the valve functions (308 a-f/h) in the connectingmicroconduits (307 a-f/h).

The aliquot applied should have a surface tension, which is ≧5 mN/m,such as ≧10 mN/m or ≧20 mN/m.

D. Unit D: Inlet Unit with Means Supporting Liquid Entrance into aMicrochannel Structure

This subaspect of the invention refers to an improvement that lowers thetime for undesired evaporation of an aliquot that has been dispensed toa microfluidic device of the same kind as the invention. The advantagesare primarily related to dispensing and/or metering nl-aliquots withinmicrofluidic devices.

The fourth sub-aspect of the invention is a microfluidic devicecomprising a microchannel structure in which there is an inlet unitpromoting liquid entrance into a microchannel structure.

The unit is illustrated in FIGS. 4 a-b. The unit comprises: (a) an inletport comprising a microcavity (401) and an inlet opening (402), and (b)an inlet conduit (403) which is positioned downstream said microcavity(401) and which communicates with the interior of the microchannelstructure.

The inner wall of the microcavity (401) comprises one or more groovesand/or projections (ridges/valleys) (404) directed towards theconnection between the inlet conduit (403) and the microcavity (401).The microcavity (401) is typically tapered (narrowing) when approachingthe inlet microconduit (403).

The main purpose of the grooves and/or the projections is to increasethe capillary suction in the inlet port. This will speed up liquidpenetration and lower the time for undesired evaporation and loss ofliquid during the dispensing operation.

The narrowing design of microcavity (401) as such assist in promotingliquid penetration and of retaining a dispensed aliquot within thecovered part of a microchannel structure.

FIG. 4 b illustrates a variant comprising a non-wetting surface break(405) in association with the rim of the inlet opening (401), primarilyat a side which is closest to the spinning axis if the inlet port islocated on a spinning substrate. This figure also illustrates a variantof unit D that comprises anti-wicking means downstream the inlet opening(401). These means may comprise changes in geometric surfacecharacteristics (406) and/or in chemical surface characteristics (407).

The projections may have a height that at maximum is equal to the depthof the microcavity (401) but may be significantly lower as long as asufficient capillary action (self-suction) is maintained in the inletport in order to draw a dispensed aliquot completely into the coveredpart of a microchannel structure.

The liquid to be introduced typically has a surface tension as discussedabove.

The width of the inlet opening is typically smaller than the width ofmicrocavity (401) as illustrated in FIGS. 4 a-b.

The inlet opening (402) may have one or more edges directed inwards theport, preferably with an n-numbered axis of symmetry perpendicular tothe opening. n is preferably an integer ≦7, such as 3, 4, 5 or 6. Seefor instance U.S. Pat. No. 4,233,029 (Eastman Kodak) and U.S. Pat. No.4,254,083 (Eastman Kodak).

Unit D is typically combined with a dispenser that is capable ofdispensing an aliquot in the nl-range to the inlet port. The dispensercan be one of the dispensers generally described elsewhere in thisspecification.

Other forces than capillary force may be used for promoting penetrationthrough the inlet port, for instance inertia force including centrifugalforce.

Microchannel structures that comprise unit D are in a preferred variantplaced on a spinnable substrate as discussed elsewhere in thisspecification.

This kind of inlet unit is particularly well adapted to receive aliquotsthat are in the form of particle suspensions.

E. Unit E: Definition of the Volume of Aliquots

In spite of the previously known devices for metering aliquots in theμl-range there is still a need for improvements, in particular withrespect to the nl-range. The reason is that uncontrolled evaporation hasa stronger influence on a smaller aliquot more compared to a largeraliquot (respect relative loss in volume). This is further accentuatedwhen a large number of aliquots are to be dispensed in sequence beforethe aliquots are further processed within a microfluidic device.

The present inventors have recognized these problems and designed avolume-metering unit (unit E) to meter primarily nl-volumes of liquids.The unit can be integrated into microchannel structures of microfluidicdevices.

The fifth subaspect of the invention thus is a microfluidic device thatcomprises a microchannel structure in which there is volume-definingunit enabling accurate metering of small volumes within a microfluidicdevice, primarily nl-volumes.

Unit E is illustrated in FIG. 5. Unit E comprises: (a) a volume-definingmicrocavity (501); (b) an inlet microconduit (502) which is connected tothe microcavity (501) via an inlet opening on the microcavity (501) (atthe joint between said microcavity and the inlet microconduit), (c) anoutlet microconduit (503) which is connected to microcavity (501) via anoutlet opening in microcavity (501) (at the joint between saidmicrocavity and the outlet microconduit), and (d) an overflowmicroconduit (504), which is connected to an overflow opening onmicrocavity (501) (at the joint between said microcavity and theoverflow microconduit).

The inlet opening and the overflow opening are typically at the samelevel on the microcavity (501). The overflow opening is at a higherlevel than the outlet opening and the volume between these two openingsdefines the volume to be metered in the volume-defining microcavity(501). The metered volume is typically in the nl-range as defined above,but may also be larger, such as ≦10 μl or ≦100 μl or ≦1000 μl.

The liquid typically has a surface tension as discussed above.

The overflow microconduit (504) is typically communicating with ambientatmosphere via an enlargement at the end of the overflow microconduit(504) (typically a waste chamber or a waste conduit (511). The jointbetween the overflow microconduit (504) and the enlargement is at alower level than both the connection between the overflow microconduit(504) and the lowest part of the volume-defining microcavity (501) (inreality the valve function (506) at the outlet opening of thevolume-defining microcavity).

The outlet microconduit (503) is used to transport a metered liquidaliquot further into the microchannel structure.

The volume-defining microcavity (501) may have different forms, forinstance comprise: (a) one or more capillaries, and (b) a downward bentstructure with one shank acting as the inlet and the other shank endingin an upward bent that can be used as the overflow microconduit, andwith the outlet microconduit (503) being joined at the lower part of thedownward bent and intended for transporting a metered aliquot furtherdownstream in the microchannel structure.

The cross-sectional area (a,) in the volume-defining microcavity (501)at the overflow opening is in preferred variants smaller than thelargest cross-sectional area (a₂) between the overflow opening and theoutlet opening (506). The ratio a₁/a₂ typically is ≦1/3, such as ≦1/10.This means a significant constriction of the microcavity (501) at thejoint between the overflow microconduit (504) and the microcavity (501),i.e., at the joint between inlet microconduit (502) and volume-definingmicrocavity (501).

The inlet microconduit (502) upstream the overflow opening typicallywidens, for instance to an inlet port (505), such as unit D.

Between the volume-defining unit and a true inlet port there may otherstructural/functional units, for instance a unit for sample treatmentsuch as for the removal of particulate materials.

Unit E may have a valve function (506,507,508) associated with at leastone of (a) the outlet opening of microcavity (501), (b) the inletmicroconduit (502) closely upstream the overflow opening, and (c) theoverflow microconduit (504), preferably its lower part such as inassociation with its joint with the waste conduit/chamber (511).

These valves may be mechanical valve or of any of the other typesdiscussed above, but is preferably an inner valve of the closing ornon-closing type with emphasis of the former.

At least one of the inlet microconduit (502), the outlet microconduit(503) and the overflow microconduit (504) may have anti-wicking means ofthe kinds defined elsewhere in this specification. The variant shown inFIG. 5 comprises anti-wicking means (509) in the inner edges of inletmicroconduit (502). The anti-wicking means stretches across thecorresponding inner walls as discussed above in general terms.

A microchannel structure comprising unit E may in its preferred variantsbe equipped with valve functions (506, 508), preferable inner valves ofthe non-closing type, and be present on a spinnable substrate asdiscussed elsewhere in this specification. If the intention is to drivethe liquid out of the overflow channel (504) before the metered aliquotis released via the outlet microconduit (503), it becomes important tohave a sufficiently large difference in radial distance (r₁) between theoverflow opening in the volume-defining microcavity (501) and the ending(512) of the overflow microconduit (504) in a waste chamber (511)relative to the difference (r₂) in radial distance between the overflowopening and the valve (506) in the outlet microconduit (503). r₁ shallbe essentially larger than r₂. This particularly applies if the valvefunction (506) in the outlet microconduit (503) is an inner non-closingvalve. By properly selecting r₁≧r₂ , e.g., r₁≧1.25r₂, or r₁≧1.5r₂, orr₁≧2r₂, or r₁≧5r₂, or r₁≧10r₂, it will be possible for the liquid in theover-flow microconduit to pass through the valve (508) at a lowerdriving force (e.g., lower spinning speed) than required for the liquidin the volume-defining microcavity to pass through the valve (506). Theoptimal relation between the two distances depends on various factors,such as width, breadth, wettability, roughness etc. of the microconduitsconcerned as well as surface tension, density et of the liquidconcerned.

A variant that may be adapted to spinnable substrates comprises adownward bent with the volume-defining microcavity being a part of thelower part of the bent. The overflow microconduit typically is connectedto one of the shanks of the downward bent and forms together with thisshank an upward bent. The upper part of the same shank vents to ambientatmosphere (inlet vent). An inlet port for sample (corresponds to 505)may be connected to the other shank of the same downward bent. The ventto ambient atmosphere may be designed with a sample/liquid inletfunction. The outlet conduit with a valve is connected to the lower partof the downward bent (corresponds to 503 and 506, respectively). Theoverflow microconduit (corresponds to 504) ends in a waste channel orwaste chamber with a valve function (corresponds to 508).

There are advantages with having the outlet opening connected to theoutlet microconduit (503) on microcavity (501) somewhat higher than thelowest part of the volume-defining microcavity. In such variants therewill be a small volume present below the outlet opening in which it willbe possibly to sediment and collect particulate materials and only flowthe supernatant that corresponds to a metered volume through the outletmicroconduit (503). Sedimenting can be assisted by the use ofcentrifugal force (spinning).

The use of unit E defines a method for introducing metered aliquots intomicrochannel structures. The method comprises the steps of: (i)providing a microchannel structure comprising unit E and an aliquothaving a larger volume than then the volume to be metered in the unit;(ii) introducing the liquid of aliquot into the unit; (iii) applying afirst driving force to move excess liquid out through the overflowmicroconduit (504) and a second driving force to move the metered volumethrough the outlet microconduit (503) into the remaining part of themicrochannel structure.

The driving force is selected as discussed above for the other unitswith preference for inertia force including centrifugal force when thesubstrate is spinnable.

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present invention, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present invention.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps. Yet, further, all patent applications andissued patents that are referenced herein are incorporated herein byreference.

1. A microfluidic device having a microchannel structure in which thereare two or more inlet ports, one or more outlet ports, and a structuralunit in communication with at least one of said ports, wherein thestructural unit comprises: a microcavity with an outlet opening; aninlet arrangement linked to the microcavity, wherein said microcavityhas a volume sufficient to contain simultaneously the aliquots to bemixed; a mixing microconduit connected to the outlet opening, whereinthe mixing microconduit is configured to enable mixing of the aliquotsof liquid that are miscible with each other and comprises one of thefollowing shapes: a) repeatedly curving in alternate directions andhaving substantially constant width, wherein the inlet arrangement isfurther defined as having at least two inlet ports; or b) at least onecavity in which the cross-sectional area increases from an inlet,reaches a maximum and then decreases towards the outlet, and a valveassociated with the mixing microconduit close to its joint to themicrocavity, wherein the valve is a surface hydrophobic break, whereinthe inner surfaces of the mixing microconduit have a wettabilitypermitting capillary force to draw liquid into the unit once the liquidhas passed the entrance of the unit.
 2. The microfluidic device of claim1, wherein the outlet opening is in the lower part of the microcavity.3. The microfluidic device of claim 1, wherein the inlet arrangement isconnected to the upper or lower part of the microcavity.
 4. Themicrofluidic device of claim 1, wherein the inlet arrangement comprisesa common inlet microconduit for several of the aliquots and/or separateinlet microconduits for individual liquid aliquots.
 5. The microfluidicdevice of claim 4, wherein at least one inlet microconduit comprises asubmicrocavity that is part of a volume-defining unit.
 6. Themicrofluidic device of claim 5 further comprising a valve between thesubmicrocavity in each inlet microconduit and the microcavity.
 7. Themicrofluidic device of claim 6, wherein the valve is an inner valve. 8.The microfluidic device of claim 6, wherein the valve is a capillaryvalve.
 9. The microfluidic device of claim 6, wherein the valve is acapillary valve comprising a hydrophobic surface break.
 10. Themicrofluidic device of claim 6, wherein the device comprises two or moreof the microchannel structure and the submicrocavity in one inletmicroconduit is part of a distribution channel that is common forseveral of the microchannel structures of said two or more microchannelstructures.
 11. The microfluidic device of claim 1, wherein themicrofluidic device is in the form of a disc and comprises two or moreof the microchannel structure.
 12. The microfluidic device of claim 1,wherein the microchannel structure is oriented from an inner position toan outer position relative to a spin axis enabling centrifugal forcecreated by spinning the device about the spin axis to drive liquidplaced in the microchannel structure through at least a part of themicrochannel structure.
 13. The microfluidic device of claim 5, whereinthe submicrocavity has a volume≦5,000 nl.
 14. The microfluidic device ofclaim 5, wherein the submicrocavity has a volume≦100 μl.
 15. Themicrofluidic device of claim 1, wherein the mixing microconduitcomprises a chain of interlinked microcavities.
 16. The microfluidicdevice of claim 1, wherein the mixing microconduit repeatedly curves inalternate directions and has substantially constant width.
 17. Themicrofluidic device of claim 1, wherein the mixing microconduitcomprises at least one cavity in which the cross-sectional areaincreases from the inlet, reaches a maximum and then decreases towardsthe outlet, with a steeper increase from the middle to the outlet thanfrom the inlet to the middle.
 18. A microfluidic device having amicrochannel structure in which there are two or more inlet ports, oneor more outlet ports, and a structural unit in communication with atleast one of said ports, wherein the structural unit comprises: amicrocavity with an outlet opening; an inlet arrangement linked to themicrocavity, wherein said microcavity has a volume sufficient to containsimultaneously the aliquots to be mixed and wherein said microcavity isconfigured to precollect the aliquots to be mixed in a mixingmicroconduit; a mixing microconduit connected to the outlet opening,wherein the mixing microconduit is configured to enable mixing of thealiquots of liquid that are miscible with each other and comprises oneof the following shapes: a) repeatedly curving in alternate directionsand having substantially constant width, wherein the inlet arrangementis further defined as having at least two inlet ports; or b) at leastone cavity in which the cross-sectional area increases from an inlet,reaches a maximum and then decreases towards the outlet, and a valveassociated with the mixing microconduit close to its joint to themicrocavity, wherein the valve is a surface hydrophobic break, whereinthe inner surfaces of the mixing microconduit have a wettabilitypermitting capillary force to draw liquid into the unit once the liquidhas passed the entrance of the unit.
 19. A microfluidic device having amicrochannel structure in which there are at least two inlet ports, oneor more outlet ports, and a structural unit in communication with atleast one of said ports, wherein the structural unit comprises: amicrocavity with an outlet opening; an inlet arrangement linked to themicrocavity, wherein said microcavity has a volume sufficient to containsimultaneously the aliquots to be mixed; a mixing microconduit connectedto the outlet opening, wherein the mixing microconduit is configured toenable mixing of the aliquots of liquid that are miscible with eachother and comprises one of the following shapes: a) repeatedly curvingin alternate directions and having substantially constant width; or b)at least one cavity in which the cross-sectional area increases from aninlet, reaches a maximum and then decreases towards the outlet, and avalve associated with the mixing microconduit close to its joint to themicrocavity, wherein the valve is a surface hydrophobic break, whereinthe inner surfaces of the mixing microconduit have a wettabilitypermitting capillary force to draw liquid into the unit once the liquidhas passed the entrance of the unit.
 20. A microfluidic device having amicrochannel structure in which there are two or more inlet ports, oneor more outlet ports, and a structural unit in communication with atleast one of said ports, wherein the structural unit comprises: amicrocavity with an outlet opening; an inlet arrangement linked to themicrocavity, wherein said microcavity has a volume sufficient to containsimultaneously the aliquots to be mixed; a mixing microconduit connectedto the outlet opening, wherein the mixing microconduit is configured toenable mixing of the aliquots of liquid that are miscible with eachother and comprises one of the following shapes: a) repeatedly curvingin alternate directions and having substantially constant width, whereinthe inlet arrangement is further defined as having at least two inletports; or b) at least one cavity in which the cross-sectional areaincreases from an inlet, reaches a maximum and then decreases towardsthe outlet, and a valve associated with the mixing microconduit close toits joint to the microcavity, wherein the valve is a surface hydrophobicbreak, wherein the inlet arrangement comprises a common inletmicroconduit for several of the aliquots and/or separate inletmicroconduits for individual liquid aliquots, wherein at least one inletmicroconduit comprises a submicrocavity that is part of avolume-defining unit, further comprising a valve between thesubmicrocavity in each inlet microconduit and the microcavity.
 21. Themicrofluidic device of claim 20, wherein the valve is an inner valve.22. The microfluidic device of claim 21, wherein the valve is acapillary valve.
 23. The microfluidic device of claim 21, wherein thevalve is a capillary valve comprising a hydrophobic surface break.